Lyme borreliosis is the leading vector-borne bacterial disease of humans in the world with over 20,000 cases reported annually in the US alone. The infectious cycle of the spirochetal agent of Lyme borreliosis, Borrelia burgdorferi, involves passage between the tick vector and mammalian host. The genetic determinants required for B. burgdorferi mammalian infectivity remain largely unknown. Recent attempts to identify the B. burgdorferi genes expressed in the mammal have been hindered by the inability to accurately model in vivo environmental signals in vitro, low spirochete loads in infected host tissues and the requirement for protein immunoreactivity. In order to identify genes uniquely expressed during mammalian infection, I propose to complete development of a B. burgdorferi in vivo genetic screen using a Recombinase-based In Vivo Expression Technology (RIVET) system. This gene discovery method directly selects for those promoters that confer mammalian infectivity by driving expression of an in vivo essential gene. Other advantages of this method are that it does not rely on isolation of spirochete RNA from infected tissues or protein antigenicity, and will detect weakly expressed genes that have been overlooked by other techniques. A B. burgdorferi genomic library will be constructed to drive transcription of a promoter-less fusion construct harboring the B. burgdorferi in vivo essential gene pncA and the recombinase gene cre. The presence of an active Borrelia promoter will induce gene expression of pncA, thereby restoring infectivity, as well as activate expression of cre, resulting in a recombination event that alters the antibiotic resistance of the spirochete. Pools of B. burgdorferi, representing subsets of the library, will be introduced into the mammalian host and the surviving spirochetes will be isolated following infection. B. burgdorferi genes that are expressed in the mammalian environment will be identified from spirochete clones that are able to infect mice and further characterized for their roles in the tick-mouse infectious cycle. These studies will provide insight into unknown genetic mechanisms of B. burgdorferi mammalian infectivity and the B. burgdorferi gene expression profile in the mammal. In addition, adaptation of RIVET technology to B. burgdorferi provides a new genetic method for in vivo gene discovery that will have broad application for all spirochetes and the different environments encountered throughout their infectious cycles. RELEVANCE: The bacteria Borrelia burgdorferi is transmitted to humans by the bite of an infected tick, resulting in Lyme borreliosis, the most common arthropod-borne bacterial disease in the world. The B. burgdorferi genes that cause this disease remain largely unknown. Identification of novel B. burgdorferi virulence factors may facilitate development of therapeutic protocols to reduce the incidence of Lyme borreliosis.